The cytochrome P450 enzymes (P450s) play an important role in metabolizing xenobiotics and certain endobiotics. The P450s exist in multiple isozymic forms which direct the metabolic flow of individual substrates. Gelboin, Pharmacol. Rev. 45: 413 (1993). Compounds which are metabolized by the cytochrome P450 enzymes include various drugs, carcinogens, mutagens, pesticides, steroids, fatty acids, bile acids and prostaglandins. Gonzalez, loc. cit. 40: 243 (1989). Cytochrome P450 metabolism of xenobiotics can result in detoxification of toxic compounds by their conjugation into excretable forms or can result in activation of compounds into metabolites that are toxic, mutagenic, or carcinogenic. Many steroids are deactivated by cytochrome P450-catalyzed oxidation.
The human cytochrome P450 3A family consists of several isozymes. Cytochome P450 3A4 is the most abundant of the P450 3A enzymes in the human liver, but its expression levels in the liver and other tissues vary. Enzymatically active human P450 3A4 has been produced from cloned cDNA expressed in a baculovirus expression system. Buters et al., Drug Metab. Dispos. 22: 688 (1994). The nucleotide sequence of cytochrome P450 3A3 is 98% identical to the P450 3A4 nucleotide sequence. Guengerich et al., "The Importance of Cytochrome P450 3A Enzymes in Drug Metabolism," in ASSESSMENT OF THE USE OF SINGLE CYTOCHROME P450 ENZYMES IN DRUG RESEARCH 161-186 (Springer Verlag 1994). P450 3A3 has been cloned and expressed, and its enzyme activity is nearly identical to that of 3A4. Schuetz, et al., Hepatology, 18:1254-62, 1993; Molowa, et al., Proc. Natl. Acad. Sci. U.S.A. 83:5311-5315, 1986. Some researchers believe that 3A3 is in fact 3A4 and the minor sequence variations between 3A3 and 3A4 are due to sequencing errors. 3A3 and 3A4 will be referred to separately throughout the specification, with the recognition that further research may demonstrate these two enzymes to actually be the same. Approximately one fourth of individuals have the P450 3A5 enzyme, and when expressed in the liver, its level is lower than that of the P450 3A4 enzyme. Id. P450 3A5 has not been characterized as well as the 3A4 enzyme. It has been shown that P450 3A5 and 3A4 have similar, but not identical, substrate specificities. Id.
Cytochrome P450 3A4 and 3A3 are very important members of the P450 class of enzymes. The human P450 3A4 and 3A3 assume an especially important place among the human P450s because they metabolize a large variety of drugs, steroids and carcinogens. Id. Cytochromes P450 3A3 and 3A4 are considered the most important P450s for a wide range of high molecular weight substrates which include many of the known clinically useful drugs, such as tranquilizers, anti-depressants, immunosuppressants and anti-cancer drugs. A partial list of the P450 3A substrates appears in Table 1 below.
TABLE 1 ______________________________________ P450 3A SUBSTRATES ______________________________________ epipodophyllotoxins sulfoxide tamoxifen cyclosporin teniposide (S)-warfarin erythromycin etoposide .alpha.-naphthoflavone benzphetamine midazolam phenanthrene triacetyloleandomycin taxol benzoa!pyrene nifedipine FL-506 sulfentamil cocaine vinblastine codeine cortisol afentanil imipramine testosterone vindesine dapsone aflatoxin B1 amiodarone aminochrysene lidocaine mephenytoin 1-nitropyrene amphetamine digitoxin alfentanil benzoa!pyrene 7, 8-diol ______________________________________
Cytochrome P450 2E1 plays a major role in the metabolism of a variety of low molecular weight compounds including environmental chemicals, and carcinogens such as short-chain dialkylnitrosamines, benzene, styrene, halomethanes, vinyl halides, ethyl carbamate, and small vinyl monomers. Guengerich et al., Chem. Res. Toxicol. 4: 168 (1991). The human 2E1 also metabolizes clinically useful drugs such as the anesthetic chlorzoxazone and the analgesic acetaminophen as well as caffeine. The rat 2E1 nucleotide sequence is 75% homologous to the human 2E1 and the amino acid sequences of rat and human are 78% homologous. See Song, et al. J. Biol. Chem., 261:16689-16697, 1986. A partial list of the P450 2E1 substrates appears in Table 2.
TABLE 2 ______________________________________ SUBSTRATES FOR 2E1 ______________________________________ Chlorzoxazone vinyl bromide Acetaminophen vinyl carbamate Acrylonitrile ethyl carbamate p-Nitrophenol CC14 N-Nitrosodimethylamine (NDMA) CHC13 N-Nitrodiethylamine (NDEA) 1, 2-Dichloropropane Enflurane CH3CC13 N-Nitrosonornicotine (NNN) Tricholordethylene 1, 1-Dimethylformamide Benzene Stryrene Eicosatetraenoic acid Caffeine N, N-Dimethylformamide 1, 3-Butadiene Ethylene dibromide Furan Ethylene dichloride p-Nitroanisole vinyl chloride Sevoflurane, Isoflurane & Methoxyflurane 1, 1, 2, 2-Tetrafluoro-1- (2, 2, 2-trifluoroethoxy)-ethane (HFE) ______________________________________
Many current methods for assessing the contribution of specific P450 enzymes to the metabolism of various chemicals are not effective. Chemical enzyme inhibitors are non-specific. Comparison of metabolism rates in different tissue preparations is not useful because this method does not distinguish between P450 isozymes. Reconstitution of catalytic activities using purified enzyme preparations does not provide information about enzyme activity in vivo. Inhibition of enzyme activity with antibodies to cytochrome P450s has also been reported, see Guengerich et al. (1991), supra, but the production of monoclonal antibodies to human P450s has been greatly hindered by the inability to obtain human P450s in amounts sufficient for immunization. Barnes et al. reported monoclonal antibodies made against human liver microsomes or semi-purified human P450s. These antibodies recognized a 53 kDa band on a Western blot, but there was no characterization of whether these antibodies recognize 3A3, 3A4, 3A5 or other human P450s. In addition, there was no investigation of inhibition of cytochrome P450 enzyme activity by these antibodies. Barnes et al., Proc. Nat'l Acad. Sci. USA 84: 4073 (1987). Beaune et al. obtained monoclonal antibodies against a mixture of purified human P450s, but the precise identity of the P450 isozymes with which these antibodies cross react is unclear. This report also did not discuss antibody inhibition of enzyme activity. Beaune et al., Biochem. Pharmacol. 34: 3547 (1985). Park et al., "Preparation and characterization of monoclonal antibodies to pregnenolone 16-.alpha.-carbonitrile inducible rat liver cytochrome P450," Biochem. Pharmacol. 35: 2859 (1986), reported monoclonal antibodies against rat P450 3A1/2. The structure and function of the rat 3A1/2 enzymes are similar, but not identical to the human P450 3A3 or 3A4 enzymes. Park et al. did not investigate antibody-mediated inhibition of rat 3A1/2 enzyme activity.
Guengerich et al., Chem. Res. Toxicol. 4: 168 (1991), reported on a polyclonal antibody against human P450 2E1 which inhibited the metabolism of 2E1 substrates in human liver microsomes. A monoclonal antibody against rat cytochrome P450 2E1 was prepared by Ko et al., Cancer Res. 47: 3101 (1987), and was found to inhibit P450 2E1-mediated metabolism of certain chemicals in rat microsomes. Ko et al. also isolated a monoclonal antibody against rat P450 2E1 which recognized P450 2E1 on a Western blot but did not inhibit enzyme activity.
Cytochrome P450s are a paradigm for multi-isozymic systems whose activity may result in metabolic products with opposing physiological and pathological consequences. The large multiplicity of P450 forms, their differing structure and function, their often poorly defined substrate and product specificity, and their heterogeneous distribution in vivo make difficult the determination of the quantitative contribution of each P450 to the metabolism of specific substrates.
A need therefore exists for reagents that can identify and inhibit certain human cytochrome P450 enzymes specifically. A thorough understanding of the different P450 isozymes, their specificity, regulation, and distribution is crucial to designing more effective drugs, evaluating the modes of action of drugs, carcinogens and environmental chemicals, and developing inhibitors of carcinogens and other toxic chemicals. Individuals sensitive to particular chemicals could be characterized based on their cytochrome P450 makeup, permitting administration of the most effective drugs and toxicity inhibitors. Monoclonal antibodies which inhibit cytochrome P450 3A3, 3A4, and 3A5 and P450 2E1 would be powerful analytical tools because the cytochrome P450 3A enzymes play an important role in metabolizing a wide variety of xenobiotics and endobiotics, including many large molecular weight compounds, and P450 2E1 plays an important role in metabolizing many small molecular weight chemicals and carcinogens. In order to produce specific monoclonal antibodies, there must be a large enough supply of purified cytochrome P450s to immunize for the production of antibodies.